System and method for analyte sampling and analysis

ABSTRACT

The invention relates to a transdermal analyte monitoring system comprising a medium adapted to interface with a biological membrane and to receive an analyte from the biological membrane and an electrode assembly comprising a plurality of electrodes, wherein the medium is adapted to react continuously with the analyte, an electrical signal is detected by the electrode assembly, and the electrical signal correlates to an analyte value. The analyte value may be the flux of the analyte through the biological membrane or the concentration of the analyte in a body fluid of a subject. The medium may comprise a vinyl acetate based hydrogel, an agarose based hydrogel, or a polyethylene glycol diacrylate (PEG-DA) based hydrogel, for example. The surface region of the electrode may comprise pure platinum. The system may include an interference filter located between the biological membrane and the electrode assembly for reducing interference in the system. The system may comprise a processor programmed to implement an error correction method that corrects for sensor drift.

The present application is a divisional of U.S. application Ser. No.11/201,334, filed Aug. 11, 2005, which is a continuation of U.S.application Ser. No. 10/974,963, filed Oct. 28, 2004, both of which arehereby incorporated by reference in their entireties. The presentapplication is related to the following patent and applications, each ofwhich is incorporated herein by reference it its entirety: U.S.application Ser. No. 09/979,096, filed Mar. 16, 2001; U.S. applicationSer. No. 09/868,442, filed Dec. 17, 1999; U.S. Provisional ApplicationNo. 60/112,953, filed Dec. 18, 1998; U.S. Provisional Application No.60/142,941, filed Jul. 12, 1999; U.S. Provisional Application No.60/142,950, filed Jul. 12, 1999; U.S. Provisional Application No.60/142,951, filed Jul. 12, 1999; U.S. Provisional Application No.60/142,975, filed Jul. 12, 1999; U.S. Pat. No. 6,190,315; and U.S.Provisional Application No. 60/070,813, filed Jan. 8, 1998.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to non-invasive sampling of body fluids,and, more particularly, to a system, method, and device for non-invasivebody fluid sampling and analysis.

2. Description of the Related Art

Diabetics frequently prick their fingers and forearms to obtain blood inorder to monitor their blood glucose concentration. This practice ofusing blood to perform frequent monitoring can be painful andinconvenient. New, less painful methods of sampling body fluids havebeen contemplated and disclosed. For example, these painless methodsinclude the use of tiny needles,

the use of iontophoresis, and the use of ultrasound to sample bodyfluid, such as blood and interstitial fluid.

It has been shown that the application of ultrasound can enhance skinpermeability. Examples of such are disclosed in U.S. Pat. No. 4,767,402,U.S. Pat. No. 5,947,921, and U.S. Pat. No. 6,002,961, the disclosures ofwhich are incorporated, by reference, in their entireties. Ultrasoundmay be applied to the stratum corneum via a coupling medium in order todisrupt the lipid bilayers through the action of cavitation and itsbioacoustic effects. The disruption of stratum comeum, a barrier totransport, allows the enhanced diffusion of analyte, such as glucose ordrugs, through, into, and out of the skin.

Transport of analytes and body fluids can be enhanced further by theaction of a motive force. These motive forces include, inter alia,sonophoretic, Iontophoretic, electromotive, pressure force, vacuum,electromagnetic motive, thermal force, magnetic force, chemomotive,capillary action, and osmotic. The use of active forces provide a meansfor obtaining fluid for subsequent analysis.

The application of a motive force before, during, and after making theskin permeable has been disclosed in U.S. Pat. No. 5,279,543, U.S. Pat.No. 5,722,397, U.S. Pat. No. 5,947,921, U.S. Pat. No. 6,002,961, andU.S. Pat. No. 6,009,343, the disclosures of which are incorporated byreference in their entireties. The purpose of using a motive force is toactively extract body fluid and its content out of the skin for thepurpose of analysis. As mentioned, active forces, such as vacuum,sonophoresis, and electrosmotic forces, can create convective flowthrough the stratum corneum. Although these forces can be used forextraction of body fluids, there are certain limitations that may applywhen the forces are applied to human skin. For example, a majorlimitation is the flow and volume of body fluid that can be transportedacross the stratum comeum. In general, high-pressure force is necessaryin order to transport fluid across an enhanced permeable area of stratumcorneum. The application of vacuum on skin for an extended period maycause physical separation of the epidermis from the dermis, resulting inbruises and blisters.

Another example of a limitation is the amount of energy that can beapplied to the skin in order to create convective flow. Extraction ofusable volume of body fluid has the potential to cause pain and skindamage with prolonged exposure to ultrasound. In a similar manner,electro-osmotic extraction of body fluid through stratum comeum has thepotential to cause skin damage due the need to use high current density.It is evident that there are limitations to the use of the mentionedextraction methods when applied to human skin.

SUMMARY OF THE INVENTION

Therefore, a need has arisen for a system, method, and device fornoninvasive body fluid sampling and analysis that overcomes these andother drawbacks of the related art.

Therefore, a need has arisen for a method of enhancing the permeabilityof a biological membrane, such as skin, buccal, and nails, for anextended period of time, and a method for extracting body fluid toperform blood, interstitial fluid, lymph, or other body fluid analytemonitoring in a discrete or continuous manner that is noninvasive andpractical.

According to one embodiment, the invention relates to a transdermalanalyte monitoring system comprising a medium adapted to interface witha biological membrane and to receive an analyte from the biologicalmembrane, wherein the medium comprises a hydrogel selected from thegroup consisting of vinyl acetate based hydrogels, agarose basedhydrogels, polyethylene glycol diacrylate (PEG-DA) based hydrogels andmixtures thereof, and an electrode assembly, wherein the medium isadapted to react continuously with the analyte, and wherein anelectrical signal is detected by the electrode assembly, and theelectrical signal correlates to an analyte value.

According to another embodiment, the invention relates to a transdermalanalyte monitoring system comprising a medium adapted to interface witha biological membrane and to receive an analyte from the biologicalmembrane, and an electrode assembly comprising a plurality ofelectrodes, wherein a surface region of at least one of the electrodeconsists essentially of pure platinum, wherein the medium is adapted toreact continuously with the analyte, and wherein an electrical signal isdetected by the electrode assembly, and the electrical signal correlatesto an analyte value.

According to another embodiment, the invention relates to a transdermalanalyte monitoring system comprising a medium adapted to interface witha biological membrane and to receive an analyte from the biologicalmembrane, an electrode assembly, and an interference filter locatedbetween the biological membrane and the electrode assembly for reducinginterference from non-target biological moieties in the transdermalanalyte monitoring system.

According to another embodiment, the invention relates to a transdermalanalyte monitoring system comprising a medium adapted to interface witha biological membrane and to receive an analyte from the biologicalmembrane, a sensor comprising an electrode assembly, the electrodeassembly comprising a plurality of electrodes, and a processorprogrammed to implement an error correction method that corrects forsensor drift, wherein the medium is adapted to react continuously withthe analyte, and wherein an electrical signal is detected by theelectrode assembly, and the electrical signal correlates to an analytevalue.

A method for non-invasive body fluid sampling and analysis is disclosed.According to one embodiment of the present invention, the methodincludes the steps of (1) identifying an area of biological membranehaving a permeability level; (2) increasing the permeability level ofthe area of biological membrane; (3) contacting the area of biologicalmembrane with a receiver; (4) extracting body fluid through and out ofthe area of biological membrane; (5) providing an external force toenhance the body fluid extraction; (6) collecting the body fluid in thereceiver; (7) analyzing the collected body fluid for the presence of atleast one analyte; and (8) providing the results of the step ofanalyzing the body fluid.

The area of biological membrane may be made permeable using ultrasoundwith controlled dosimetry. Extraction of body fluid may be performed onthe area exposed to ultrasound using osmotic transport. The body fluidmay be collected using a receiver. The receiver may be attached to thebiological membrane in a form of a patch, a wearable reservoir, amembrane, an absorbent strip, a hydrogel, or an equivalent. The receivermay be analyzed for the presence of various analytes indicative of bloodanalytes. The analysis may comprise the use of electrochemical,biochemical, optical, fluorescence, absorbance, reflectance, Raman,magnetic, mass spectrometry, infra-red (IR) spectroscopy measurementmethods and combinations thereof. The receiver may also be attached to asecondary receiver where the concentration of analyte in the secondaryreceiver is continuously maintained substantially lower than that in thebody fluid so the chemical concentration driving force between bodyfluid and secondary receiver is maximized. This may be achieved bychemical reaction or volume for dilution or similar means. In oneembodiment, the receiver and the secondary receiver may operate ondifferent principles (e.g., osmosis, dilution, etc.). In anotherembodiment, the receivers may operate on the same principle.

A system for non-invasive body fluid sampling and analysis is disclosed.According to one embodiment of the present invention, the systemincludes a controller that controls the generation of ultrasound; anultrasonic applicator that applies the ultrasound to an area ofbiological membrane; a receiver that contacts the area of biologicalmembrane and receives body fluid through and out of the area ofbiological membrane; and a meter that interacts with the receiver anddetects the presence of at least one analyte in the body fluid in thereceiver. The receiver may include a membrane and a medium, such as ahydrogel, a fluid, or a liquid, that is contained within the membrane.

A method for noninvasive body fluid sampling and analysis is disclosed.According to one embodiment of the present invention, the methodincludes the steps of (1) enhancing a permeability level of an area ofbiological membrane; (2) attaching a receiver to the area of biologicalmembrane; (3) extracting an analyte through and out of the area ofbiological membrane; (4) collecting the body fluid in the receiver; and(5) determining a concentration of at least one analyte in the bodyfluid.

A device for noninvasive body fluid sampling and analysis is disclosed.According to one embodiment of the present invention, the deviceincludes a receiver that is attached to an area of biological membranewith an enhanced permeability and receives body fluid through and out ofthe area of biological membrane, and a wearable meter that detects thepresence of at least one analyte in the received body fluid andindicates a concentration of that analyte. The receiver may include amembrane and a medium, such as a hydrogel, a fluid, or a liquid, that iscontained in the membrane. The meter may include a processor and adevice that detects the presence of the analyte. The detecting devicemay include an electrochemical detector; a biochemical detector; afluorescence detector; an absorbance detector; a reflectance detector; aRaman detector; a magnetic detector; a mass spectrometry detector; an IRspectroscopy detector; and combinations thereof.

According to one embodiment of the present invention, osmotic forces maybe used to sample body fluid from and through a biological membrane inan on-demand manner. The osmotic agent in solution, gel, hydrogel, orother form may be applied to the ultrasound-treated biological membraneusing a receiver, such as a thin liquid reservoir, whenever theconcentration of an analyte needs to be determined for diagnosis andmonitoring. The receiver may be attached to the biological membraneusing an adhesive. The receiver may be attached to the biologicalmembrane for a brief duration. The solution in the receiver may besubsequently removed and analyzed for the presence of analytes. In oneembodiment, the receiver may be constructed in the form of a patch. Thereceiver may contain a hydrogel and osmotic agent. The receiver maycombine the osmotic agent and the chemical reagents to detect thepresence of the analyte. The reagents may allow the use ofelectrochemical, biochemical, optical, fluorescence, absorbance,reflectance, Raman, magnetic, mass spectrometry, infrared (IR)spectroscopy measurement methods and combinations thereof to beperformed on the receiver.

In another embodiment, osmotic forces may be used to sample body fluidfrom or through a biological membrane in a periodic or a continuousmanner. The osmotic agent in solution form may be applied to theultrasound-treated biological membrane using a thin receiver, such as athin liquid reservoir, whenever the concentration of analyte needs to bedetermined for diagnosis and monitoring. The receiver may be attached tobiological membrane using an adhesive. In one embodiment, the receivermay be constructed in the form of a patch. The receiver may contain ahydrogel that contains the osmotic agent. The receiver may contain meansfor manipulating the intensity and duration of the osmotic force. Theintensity of the osmotic force may be manipulated using electric fieldforces, magnetic field forces, electromagnetic field forces, biochemicalreactions, chemicals, molarity adjustment, adjusting solvents, adjustingpH, ultrasonic field forces, electro-omostic field forces, iontophoreticfield forces, electroporatic field forces and combinations thereof. Theduration of the osmotic force may be manipulated using electric fieldforces, magnetic field forces, electromagnetic field forces, biochemicalreactions, chemicals, molarity adjustment, adjusting solvents, adjustingpH, ultrasonic field forces, electroomostic field forces, iontophoreticfield forces, electroporatic field forces and combinations thereof. Thereceiver may combine the osmotic agent and the biochemical reagents todetect the presence of the analyte. The reagents may allow the use ofelectrochemical, biochemical, optical, fluorescence, absorbance,reflectance, Raman, magnetic, mass spectrometry, IR spectroscopymeasurement methods and combinations thereof to be performed on thereceiver. The receiver may also be removed periodically for detection.

In one embodiment, the intensity, duration, and frequency of exposure ofbiological membrane to osmotic forces may be manipulated by using anelectric current to cause a change in the concentration of the osmoticagent that is in contact with the ultrasound-exposed biologicalmembrane. The osmotic agent may be a multi-charged agent that candissociate into several charged species. These charged species may betransported using electric field forces. A membrane may be used toisolate the charged species. The charged species freely diffuse andcombine upon removal of the electric field force.

In one embodiment, the intensity, duration, and frequency of exposure ofbiological membrane to osmotic forces may be manipulated by using activeforces to cause a change in the concentration of the osmotic agent thatis in contact with the ultrasound-exposed biological membrane. Theosmotic agent may be a neutral charge agent. The agent may betransported using a variety of field forces. The field force depends onthe constitutive and colligative properties of the chosen agent. Thefield force generates a force necessary to move the osmotic agent towardand away from the biological membrane surface. The movement of theosmotic agent modulates the periodic and continuous extraction of bodyfluid through the stratum comeum.

In one embodiment, the intensity, duration, and frequency of exposure ofbiological membrane to osmotic forces may be manipulated by changing theconcentration of the osmotic agent that is in contact with theultrasound-exposed biological membrane. Manipulating the volume of thesolvent and the volume of the hydrogel containing the osmotic agent maycause a change in the concentration of the osmotic agent. The volume ofthe hydrogel can be changed by constructing a hydrogel wherein itsvolume is sensitive to the concentrations of molecules that can diffuseinto the gel. One example is a hydrogel constructed to be sensitive tothe molecule glucose. The hydrogel volume can also be changed bymanipulating its temperature and by changing the pH of the gel.

A receiver that is attached to an area of biological membrane with anenhanced permeability and receives body fluid through and out of thearea of biological membrane is disclosed. According to one embodiment ofthe present invention, the receiver includes a first grid; a mediumlayer comprising at least one agent; a membrane that induces aconcentration gradient barrier for the at least one agent; a countergrid; an oxidase layer; a detection layer; and a voltage source thatprovides a potential difference between the first grid and the countergrid. The body fluid, which may include blood, interstitial fluid,analyte, and lymph, may flow out of, or through, the biologicalmembrane, to the detector layer via the first grid, the counter grid,and the oxidase layer.

It is a technical advantage of the present invention that a system,method, and device for non-invasive sampling and analysis of body fluidsis disclosed. It is another technical advantage of the present inventionthat a concentration of an analyte may be measured continuously orperiodically.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, the objectsand advantages thereof, reference is now made to the followingdescriptions taken in connection with the accompanying drawings inwhich:

FIG. 1 is a flowchart depicting a method for non-invasive body fluidsampling according to one embodiment of the present invention;

FIG. 2 depicts a device for controlled application of ultrasound to abiological membrane to enhance the permeability of the biologicalmembrane according to one embodiment of the present invention;

FIG. 3 depicts the components to perform discrete extraction andmeasurement of body fluid to infer analyte concentrations according toone embodiment of the present invention;

FIG. 4 depicts the components to perform continuous extraction andmeasurement of body fluid to infer analyte concentrations according toone embodiment of the present invention;

FIG. 5 depicts an approach to periodic monitoring of an analyte byperforming periodic osmotic extractions of body fluid according to oneembodiment of the present invention;

FIG. 6 depicts the components of a wearable extraction chamber accordingto one embodiment of the present invention;

FIG. 7 depicts a graph of glucose flux versus blood glucoseconcentration according to one embodiment of the present invention;

FIG. 8 depicts a flow chart of a method for controlled enhancement oftransdermal delivery according to one embodiment of the presentinvention;

FIG. 9 depicts an apparatus for performing continuous transdermalanalyte monitoring according to one embodiment of the present invention;

FIG. 10 is a drawing of the sensor body shown in FIG. 9 from a firstview;

FIG. 11 is a drawing of the apparatus shown in FIG. 9 from a secondview;

FIG. 12 shows the signal response versus glucose concentration forvarious hydrogels;

FIG. 13 shows the signal response versus glucose concentration for pureplatinum versus platinized carbon as the working electrode;

FIG. 14 shows the current-time profiles of a glucose sensor respondingto the addition of hydrogen peroxide using platinum and platinizedcarbon as the working electrode;

FIG. 15 shows the sensor response to hydrogen peroxide (HP) overacetominophen (AM) and hydrogen peroxide over uric acid (UA) for sensorswith and without a Nafion interference filter;

FIG. 16 shows a Clark Error Grid in the absence of an error correctionmethod according to one embodiment of the invention;

FIG. 17 shows a Clark Error Grid after the application of an errorcorrection method according to an embodiment of the invention;

FIG. 18 shows the absorbance spectrum of a standard glucose oxidasesolution before and after incorporation into a PEGDA 3.4K gel;

FIG. 19 shows the signal response to glucose of glucose oxidase (GOx)loaded PEG gels of varying molecular weight;

FIG. 20 shows signal response to glucose of 3.4K PEG hydrogel loadedwith varying concentrations of GOx;

FIG. 21 shows raw data of the potentiometric signals elicited from PEGDAhydrogels with GOx incorporated in the gel formulation prior tophotocrosslinking;

FIG. 22 shows the change in signal between GOx-presoaked versuspre-incorporated hydrogels at different thickness and compositions(PEGDA-nVP, PEGDA);

FIG. 23(a) shows blood glucose versus time utilizing an embodiment ofthe continuous transdermal analyte monitoring system;

FIG. 23(b) shows a correlation plot of electrode signal in nanoampsversus blood glucose for an embodiment of the invention;

FIG. 24 shows patient data for participants in a clinical study;

FIG. 25 shows a noisy data set from the clinical study;

FIG. 26 shows another data set from the clinical study;

FIG. 27 shows a Clark Error Grid for sensor data from the clinical studyaccording to one embodiment of the invention; and

FIG. 28 shows a Clark Error Grid for sensor data from the clinical studyaccording to another embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The preferred embodiments of the present invention and their advantagesare best understood by referring to FIGS. 1 through 28 of the drawings,like numerals being used for like and corresponding parts of the variousdrawings.

As used herein, the term “body fluid” may include blood, interstitialfluid, lymph, and/or analyte. In addition, as used herein, the term“biological membrane” may include tissue, mucous membranes and comifiedtissues, including skin, buccal, and nails. Further, as used herein, theterm “force” may also include force gradients.

Although the present invention may be described in conjunction withhuman applications, veterinary applications are within the contemplationand the scope of the present invention.

Referring to FIG. 1, a flowchart depicting a method for non-invasivebody fluid sampling and analysis according to one embodiment of thepresent invention is provided. In step 102, the permeability of an areaof biological membrane is enhanced. In one embodiment, the area ofbiological membrane may be located on the volar forearm of a mammaliansubject. In another embodiment, the area of biological membrane may belocated on a thigh of a mammalian subject. In yet another embodiment,the area of biological membrane may be located on the abdomen. In stillanother embodiment, the area of biological membrane may be located onthe back. Other body locations may also be used.

In general, several techniques may be used to enhance the permeabilityof the biological membrane, such as creating physical micropores,physically disrupting the lipid bilayers, chemically modifying the lipidbilayers, physically disrupting the stratum comeum, and chemicallymodifying the stratum corneum. The creation of micropores, or thedisruption thereof, may be achieved by physical penetration using aneedle, a microneedle, a silicon microneedle, a laser, a laser incombination with an absorbing dye, a heat source, an ultrasonic needle,an ultrasonic transducer, cryogenic ablation, RF ablation,photo-acoustic ablation, and combinations thereof.

In a preferred embodiment, ultrasound may be applied to the area ofbiological membrane to enhance its permeability. Ultrasound is generallydefined as sound at a frequency of greater than about 20 kHz.Therapeutic ultrasound is typically between 20 kHz and 5 MHz. Nearultrasound is typically about 10 kHz to about 20 kHz. It should beunderstood that in addition to ultrasound, near ultrasound may be usedin embodiments of the present invention.

In general, ultrasound, or near ultrasound, is preferably applied to thearea of biological membrane at a frequency sufficient to causecavitation and increase the permeability of the biological membrane. Inone embodiment, ultrasound may be applied at a frequency of from about10 kHz to about 500 kHz. In another embodiment, ultrasound may beapplied at a frequency of from about 20 kHz to about 150 kHz. In yetanother embodiment, the ultrasound may be applied at 50 kHz. Otherfrequencies of ultrasound may be applied to enhance the permeabilitylevel of the biological membrane.

In one embodiment, the ultrasound may have an intensity in the range ofabout 0 to about 100 watt/cm², and preferably in the range of 0 to about20 watt/cm². Other appropriate intensities may be used as desired.

Techniques for increasing the permeability of a biological membrane aredisclosed in U.S. Pat. No. 6,190,315 to Kost et al., the disclosure ofwhich is hereby incorporated by reference in its entirety.

In step 104, body fluid is extracted through or out of the area ofbiological membrane. In one embodiment, an external force, such as anosmotic force, may assist in the extraction. In one embodiment, theosmotic force may be controlled before, during, and after thepermeability of the biological membrane is enhanced.

In one embodiment, the osmotic force may be generated by the applicationof an osmotic agent to the area of biological membrane. The osmoticagent may be in the form of an element, a molecule, a macromolecule, achemical compound, or combinations thereof. The osmotic agent may alsobe combined with a liquid solution, a hydrogel, a gel, or an agenthaving a similar function.

In step 106, the magnitude, intensity, and duration of the externalforce may be regulated by at least one additional first energy and/orforce. In one embodiment, the first additional energy and/or force maybe applied to control and regulate the movement and function of theosmotic agent for extraction of body fluid through and out of thebiological membrane. The first additional energy and/or force may beprovided in the form of heat, a temperature force, a pressure force, anelectromotive force, a mechanical agitation, ultrasound, iontophoresis,an electromagnetic force, a magnetic force, a photothermal force, aphotoacoustic force, and combinations thereof. The effect of an electricfield and ultrasound on transdermal drug delivery is disclosed in U.S.Pat. No. 6,041,253, the disclosure of which is incorporated, byreference, in its entirety.

In one embodiment, if the first additional energy and/or force isprovided by ultrasound, the frequency of the ultrasound may be providedat a different frequency than the frequency used to enhance thepermeability of the biological membrane. In one embodiment, thefrequency of the first additional energy/force ultrasound may be higherthan the frequency of the permeability enhancing ultrasound.

In step 108, the body fluid may be collected in a receiver. In oneembodiment, the receiver may be contacted with the biological membranein a form of a patch, a wearable reservoir, a membrane, an absorbentstrip, a hydrogel, or a structure that performs an equivalent function.Other types and configurations of receivers may be used.

In one embodiment, the receiver may be provided with a secondaryreceiver having an analyte concentration that is continuously maintainedto be substantially lower than the analyte concentration in the bodyfluid, so the chemical concentration driving force between body fluidand secondary receiver is maximized. This may be achieved by chemicalreaction or volume for dilution or similar means.

In one embodiment, a second external energy/force may be applied betweenthe first receiver and the secondary receiver. In one embodiment, thesecond external energy/force may be different (e.g., a different type ofexternal force) from the first external energy/force. In anotherembodiment, the second external energy/force may be the same (e.g., thesame type of external force) as the first external energy/force. Thefirst and second external energy/force may vary in type, duration, andintensity, and may be controlled through different additional energyand/or forces.

In step 110, the collected body fluid may be analyzed. In oneembodiment, the analysis may include the use of appropriate methods,such as electrochemical, biochemical, optical, fluorescence, absorbance,reflectance, Raman, magnetic, mass spectrometry, infra-red (IR)spectroscopy measurement, and combinations thereof.

In one embodiment, multiple analytes may be analyzed simultaneously, inparallel, or in series. The results from these multiple analyses may beused in combination with algorithms, for example, to increase theaccuracy, or precision, or both, of the analysis and measurements.

In one embodiment, the receiver may be removed from contact with thebiological membrane in order to analyze the collected body fluid. Inanother embodiment, the receiver may remain in contact with thebiological membrane as the collected body fluid is analyzed.

Referring to FIG. 2, a device for the controlled application ofultrasound to biological membrane to enhance the permeability of abiological membrane according to one embodiment of the present inventionis shown. Device 200 includes controller 202, which interfaces withultrasound applicator 204 by any suitable means, such as a cable.Controller 202 controls the application of ultrasound to the area ofbiological membrane. In one embodiment, ultrasound or near ultrasoundhaving an intensity in the range of about 0 to about 20 watt/cm² may begenerated by controller 202 and ultrasound applicator 204. In oneembodiment, the ultrasound may have a frequency of about 20 kHz to about150 kHz. In another embodiment, the ultrasound may have a frequency of50 kHz. Other ultrasound frequencies may also be used.

In addition, controller 202 may include a display, such as a LCD or aLED display, in order to convey information to the user as required.Controller 202 may also include a user interface as is known in the art.

Ultrasound applicator 204 may be provided with cartridge 206, whichcontains ultrasound coupling solution 208. Cartridge 206 may be made ofany material, such as plastic, that may encapsulate ultrasound couplingsolution 208. Suitable ultrasound coupling solutions 208 include, butare not limited to, water, saline, alcohols including ethanol andisopropanol (in a concentration range of 10 to 100% in aqueoussolution), surfactants such as Triton X-100, SLS, or SDS (preferably ina concentration range of between 0.001 and 10% in aqueous solution),DMSO (preferably in a concentration range of between 10 and 100% inaqueous solution), fatty acids such as linoleic acid (preferably in aconcentration range of between 0.1 and 2% in ethanol-water (50:50)mixture), azone (preferably in a concentration range of between 0.1 and10% in ethanol-water (50:50) mixture), polyethylene glycol in aconcentration range of preferably between 0.1 and 50% in aqueoussolution, histamine in a concentration range of preferably between 0.1and 100 mg/ml in aqueous solution, EDTA in a concentration range ofpreferably between one and 100 mM, sodium hydroxide in a concentrationrange of preferably between one and 100 mM, sodium octyl sulfate,N-tauroylsarcosine, octyltrimethyl ammoniumbromide, dodecyltrimethylammoniumbromide, tetradecyltrimethyl ammoniumbromide, hexadecyltrimethylammoniumbromide, dodecylpyridinium chloride hydrate, SPAN 20, BRIJ 30,glycolic acid ethoxylate 4-ter-butyl phenyl ether, IGEPAL CO-210, andcombinations thereof.

In one embodiment, the coupling medium may also include a chemicalenhancer. Transport enhancement may be obtained by adding capillarypermeability enhancers, for example, histamine, to the coupling medium.The concentration of histamine in the coupling medium may be in therange of between 0.1 and 100 mg/ml. These agents may be delivered acrossthe biological membrane during application of ultrasound and may causelocal edema that increases local fluid pressure and may enhancetransport of analytes across the biological membrane. In addition, theoccurrence of free fluid due to edema may induce cavitation locally soas to enhance transport of analytes across the biological membrane.

In one embodiment, cartridge 206 may be pierced when inserted intoultrasound applicator 204, and ultrasound coupling solution 208 may betransferred to a chamber (not shown).

A target identifying device, such as target ring 210, may be attached tothe area of biological membrane that will have its permeabilityincreased. Target ring 210 may be attached to the area of biologicalmembrane by a transdermal adhesive (not shown). In one embodiment,target ring 210 may have the transdermal adhesive pre-applied, and maybe disposed after each use. In another embodiment, target ring 210 maybe reusable.

Target ring 210 may be made of any suitable material, including plastic,ceramic, rubber, foam, etc. In general, target ring 210 identifies thearea of biological membrane for permeability enhancement and body fluidextraction. In one embodiment, target ring 210 may be used to holdreceiver 214 in contact with the biological membrane after thepermeability of the biological membrane has been increased.

In one embodiment, target ring 210 may be used to monitor thepermeability level of the biological membrane, as disclosed in PCTInternational Patent Appl'n Ser. No. PCT/US99/30067, entitled “Methodand Apparatus for Enhancement of Transdermal Transport,” the disclosureof which is incorporated by reference in its entirety. In such anembodiment, target ring 210 may interface with ultrasound applicator204.

Ultrasound applicator 204 may be applied to target ring 210 andactivated to expose ultrasound coupling solution 208 to the biologicalmembrane. Controller 202 controls ultrasound applicator 204 to transmitultrasound through ultrasound coupling solution 208. During ultrasoundexposure, controller 202 may monitor changes in biological membranepermeability, and may display this information to the user.

Controller 202 may cease, or discontinue, the application of ultrasoundonce a predetermined level of biological membrane permeability isreached. This level of permeability may be preprogrammed, or it may bedetermined in real-time as the ultrasound is applied. The predeterminedlevel of permeability may be programmed for each individual due tobiological membrane differences among individuals.

After the predetermined level of permeability is reached, ultrasoundcoupling solution 208 may be vacuated from chamber (not shown) intocartridge 206, which may then be discarded. In another embodiment,ultrasound coupling solution 208 may be vacuated into a holding area(not shown) in ultrasound applicator 204, and later discharged.Ultrasound applicator 204 may then be removed from target ring 210.

Referring to FIG. 3, an device for the analysis of body fluid accordingto one embodiment of the present invention is provided. Receiver 214 maybe placed into target ring 210 to perform a discrete, or on-demand,extraction of body fluid through and/or out of the biological membrane.Receiver 214 may contain a medium, such as a hydrogel layer, thatincorporates an osmotic agent. In one embodiment, the hydrogel may beformulated to contain phosphate buffered saline (PBS), with the salinebeing sodium chloride having a concentration range of about 0.01 M toabout 10 M. The hydrogel may be buffered at pH 7. Other osmotic agentsmay also be used in place of, or in addition to, sodium chloride.Preferably, these osmotic agents are non-irritating, non-staining, andnon-immunogenic. Examples of such osmotic agents include, inter alia,lactate and magnesium sulfate.

In another embodiment, receiver 214 may include a fluid or liquidmedium, such as water or a buffer, that is contained within asemi-permeable membrane. Receiver 214 may also include a spongymaterial, such as foam.

Receiver 214 may be applied to the biological membrane to contact theultrasound exposed biological membrane. In one embodiment, receiver 214may be applied to the biological membrane for a time period sufficientto collect an amount of body fluid sufficient for detection. In anotherembodiment, receiver 214 may be applied to the biological membrane for asufficient time period to collect a predetermined amount of body fluid.In yet another embodiment, receiver 214 may be applied to the biologicalmembrane for a predetermined time. In one embodiment, the contactbetween receiver 214 and the biological membrane may last for 15 minutesor less. In another embodiment, the contact between receiver 214 and thebiological membrane may last for 5 minutes or less. In still anotherembodiment, the contact between receiver 214 and the biological membranemay last for 2 minutes or less. The actual duration of contact maydepend on the sensitivity of the detection method used for analysis.

In one embodiment, the medium of receiver 214 may contain at least onereagent (not shown) in order to detect the presence of certain analytesin the body fluid that has been extracted from or through the biologicalmembrane. In one embodiment, the hydrogel layer of receiver 214 maycontain the reagents, and the reagents may be attached to the hydrogelby ionic and/or covalent means, or may be immobilized by gel entrapment.The reagents may also be arranged as an adjacent layer to the hydrogelwherein the analyte from the body fluid that has been extracted into thehydrogel can diffuse into and react to generate by-products. Theby-products may then be detected using electrochemical, biochemical,optical, fluorescence, absorbance, reflectance, Raman, magnetic, massspectrometry, IR spectroscopy measurement methods and combinationsthereof.

The detection methods may be performed by meter 212. Meter 212 mayinclude a processor (not shown) and a display, such as an LCD display.Other suitable displays may be provided.

In one embodiment, meter 212 may provide an interface that allowsinformation be downloaded to an external device, such as a computer.Such an interface may allow the connection of interface cables, or itmay be a wireless interface.

Meter 212 may be configured to determine body fluid glucoseconcentration by incorporating glucose oxidase in the medium of receiver214. In one embodiment, glucose from extracted body fluid may react withglucose oxidase to generate hydrogen peroxide. Hydrogen peroxide may bedetected by the oxidation of hydrogen peroxide at the surface ofelectrodes incorporated into receiver 214. The oxidation of hydrogenperoxide transfers electrons onto the electrode surface which generatesa current flow that can be quantified using a potentiostat, which may beincorporated into meter 212. A glucose concentration proportional to theconcentration of hydrogen peroxide may be calculated, and the result maybe reported to the user via a display. Various configurations ofelectrodes and reagents, known to those of ordinary skill in the art,may be incorporated to perform detection and analysis of glucose andother analytes.

Meter 212 may also be configured to simultaneously measure theconcentration of an analyte, such as glucose, where the body fluidconcentration is expected to fluctuate, and an analyte, like creatinineor calcium, where the body fluid concentration is expected to remainrelatively stable over minutes, hours, or days. An analyteconcentration, which may be determined by an algorithm that takes intoaccount the relative concentrations of the fluctuating and the morestable analyte, may be reported to the user via a display.

In another embodiment, meter 212 may analyze multiple analytessimultaneously, in parallel, or in series. The results from thesemultiple analyses may be used in combination with algorithms, forexample, to increase the accuracy, or precision, or both, of theanalysis and measurements.

Receiver 214 may be discarded after the extraction and measurementsteps. In another embodiment, receiver 214 may be reused. In oneembodiment, receiver 214 may be cleaned, sanitized, etc. before it maybe reused. Various configurations of electrodes and reagents, known tothose of ordinary skill in the art, may be incorporated to performdetection and analysis of glucose and other analytes.

Referring to FIG. 4, a device for the continuous extraction and analysisof body fluid to infer analyte concentrations according to anotherembodiment of the present invention is provided. As shown in the figure,a biological membrane site on the forearm, the abdomen, or thigh may beexposed to ultrasound; other biological membrane sites, such as those onthe back, may also be used. Receiver 402, which may be similar toreceiver 214, may contact the ultrasound exposed biological membranesite to perform continuous extraction of body fluid. In one embodiment,receiver 402 may contain a medium, such as a hydrogel layer, that mayincorporate an osmotic agent, such as sodium chloride. The hydrogel isformulated to contain phosphate buffered saline (PBS), with the salinebeing sodium chloride in the concentration range of 0.01 M to 10 M. Thehydrogel may be buffered at pH 7.

Other osmotic agents may also be used in place of, or in addition to,sodium chloride. These osmotic agents are preferably non-irritating,non-staining, and non-immunogenic. Examples of these other osmoticagents may include, inter alia, lactate and magnesium sulfate. Receiver402 may be applied to contact the ultrasound exposed biologicalmembrane. In one embodiment, the duration of this contact may be 12-24hours, or more. In another embodiment, other durations of contact,including substantially shorter durations, and substantially longerdurations, may be used as desired.

In another embodiment, receiver 402 may include a fluid or liquidmedium, such as water or a buffer, that is contained within asemi-permeable membrane. Receiver 402 may also include a spongymaterial, such as foam.

In one embodiment, the medium of receiver 402 may contain at least onereagent (not shown) that detects the presence of analytes in the bodyfluid that has been extracted thorough and out of the biologicalmembrane. In one embodiment, the hydrogel layer of receiver 402 maycontain reagents that may be attached by ionic and covalent means to thehydrogel, or may be immobilized by gel entrapment. The reagents may alsobe arranged as an adjacent layer to the hydrogel wherein the analytefrom the body fluid that has been extracted into the hydrogel maydiffuse into and react to generate by-products. The by-products may bedetected using electrochemical, biochemical, optical, fluorescence,absorbance, reflectance, Raman, magnetic, mass spectrometry, IRspectroscopy measurement methods and combinations thereof.

The detection methods and results may be performed and presented to theuser by meter 404, which may be similar in function to meter 212,discussed above. In one embodiment, meter 404 may be wearable. Forexample, as depicted in the figure, meter 404 may be worn in a mannersimilar to the way a wristwatch is worn. Meter 404 may also be worn on abelt, in a pocket, etc.

Meter 404 may incorporate power and electronics to control the periodicextraction of body fluid, to detect analyte, and to present the analyteconcentration in a continuous manner. Meter 404 may contain electronicsand software for the acquisition of sensor signals, and may performsignal processing, and may store analysis and trending information.

In one embodiment, meter 404 may provide an interface that allowsinformation be downloaded to an external device, such as a computer.Such an interface may allow the connection of interface cables, or itmay be a wireless interface.

Meter 404 may be configured to determine body fluid glucoseconcentration by incorporating glucose oxidase in the medium. In oneembodiment, glucose from extracted body fluid may react with glucoseoxidase to generate hydrogen peroxide. Hydrogen peroxide may be detectedby the oxidation of hydrogen peroxide at the surface of electrodesincorporated into receiver 402. The oxidation of hydrogen peroxidetransfers electrons onto the electrode surface which generates a currentflow that can be quantified using a potentiostat, which may beincorporated into meter 404. A glucose concentration proportional to theconcentration of hydrogen peroxide may be calculated and the result maybe reported to the user via a display. Various configurations ofelectrodes and reagents, known to those of ordinary skill in the art,may be incorporated to perform detection and analysis of glucose andother analytes.

In one embodiment, meter 404 may also be configured to simultaneouslymeasure concentration of an analyte, such as glucose, where the bodyfluid concentration is expected to fluctuate, and an analyte, likecreatinine or calcium, where the body fluid concentration is expected toremain relatively stable over minutes, hours, or days. An analyteconcentration, which may be determined by an algorithm that takes intoaccount the relative concentrations of the fluctuating and the morestable analyte, may be reported to the user via a display.

In another embodiment, meter 404 may analyze multiple analytessimultaneously, in parallel, or in series. The results from thesemultiple analyses may be used in combination with algorithms, forexample, to increase the accuracy, or precision, or both, of theanalysis and measurements.

In another embodiment, receiver 402 may be removed from contact with thebiological membrane for analysis by meter 404. Receiver 402 may be putin contact with the biological membrane after such analysis.

Meter 404 may provide analyte readings to the user in a periodic or acontinuous manner. For example, in one embodiment, in continuousmonitoring of the analyte glucose, glucose concentration may bedisplayed to the user every 30 minutes, more preferably every 15minutes, most preferable every 5 minutes, or even more frequently. Inanother embodiment, the glucose concentration may be displayedcontinuously. The period may depend on the sensitivity and method ofanalyte detection. In continuous glucose monitoring, in one embodiment,glucose detection may be performed by an electrochemical method usingelectrodes and reagents incorporated into receiver 402 and detection andanalysis performed by meter 404. During the measurement period, osmoticextraction of body fluid may be performed continuously by the hydrogellayer of receiver 402. Body fluid may accumulate in the hydrogel ofreceiver 402. Glucose in body fluid diffuses to react with glucoseoxidase and is converted into hydrogen peroxide. The hydrogen peroxideis consumed by poising the working electrode with respect to a referenceelectrode. During the resting period, hydrogen peroxide accumulates andis consumed or destroyed before the measuring period. The magnitude ofthe working potential can be applied to rapidly consume the build up ofhydrogen peroxide.

Referring to FIG. 5, an approach to periodic monitoring of an analyte byperforming periodic osmotic extractions of body fluid according toanother embodiment of the present invention is shown. The osmoticextraction intensity and frequency may be manipulated by using anosmotic agent that dissociates into multiple charged species, and anelectrical potential may be used to move the concentration of chargestoward and away from biological membrane surface 550. Receiver 500 mayinclude grid, mesh, or screen 504; medium 506, which may be a hydrogellayer; membrane 508; counter grid, mesh, or screen 510; oxidase layer512; and detection layer 514. Grid 504 and counter grid 510 may beconnected to voltage source 516. Membrane 508 may be a semi-permeablemembrane that is used to induce a concentration gradient barrier for theosmotic agent contained in medium 506. The preferable osmotic agent maycontain negative and positive species or counter ions. Manipulating theconcentration of charged species at the boundary adjacent to the stratumcorneum of the ultrasound-exposed biological membrane may provideperiodic extraction of body fluid.

In one embodiment, receiver 500 may make contact with the skin thoughcontact medium 502, which may be a hydrogel, or other suitable medium.

The concentration of the charged species may be manipulated by applyinga potential difference between grid 504 and counter grid 510 usingvoltage source 516. In one embodiment, the potential difference may beof a magnitude that is sufficient to manipulate the osmotic agent. Thepolarity of the grid may also be changed to transport charges toward andaway from biological membrane surface 550. Grid 504 and counter grid 510may be configured with optimum porosity as to allow body fluid and/oranalyte to travel out of stratum corneum, through grid 504, through grid510, and into oxidase layer 512, and ultimately to detection layer 514.Oxidase layer 512 may be used with an appropriate catalyst, or enzyme,to confer specificity of analyte detection. Detection layer 514 mayinclude working and reference electrodes (not shown) that allow for thedetection of the by-products of oxidase layer 512 to quantify theconcentration of the desired analyte of detection.

EXAMPLE 1

The following example does not limit the present invention in any way,and is intended to illustrate an embodiment of the present invention.

The following is a description of experiments which implemented painlessextraction, collection, and analysis of body fluid to determine bodyfluid glucose concentration in a human using a hyperosmotic extractionfluid and comparing this condition with iso-osmotic extraction fluid, inaccordance with one embodiment of the present invention. Although bodyfluid glucose concentration serves as an example to demonstratefeasibility, other analytes are within the contemplation of the presentinvention. In addition, multiple analytes may be measured and/oranalyzed simultaneously, in parallel, or in series, and results fromthese multiple measurements may be used in combination with algorithms,for example, to increase the accuracy or precision or both ofmeasurements. As may be recognized by one of ordinary skill in the art,these steps may be automated and implemented with the device describedabove.

Four sites on the volar forearm of a human volunteer were treated withultrasound using the device described in FIG. 2. The ultrasoundtransducer and its housing were placed on the volar forearm of thevolunteer with enough pressure to produce a good contact between theskin and the outer transducer housing, and to prevent leaking. The areasurrounding the transducer was then filled with a coupling medium ofsodium dodecyl sulfate and silica particles in phosphate-buffered saline(PBS). Ultrasound was briefly applied (5-30 s), the transducer apparatuswas removed from the biological membrane, and the skin was rinsed withtap water and dried.

FIG. 6 describes the components of wearable extraction chamber 600. Fourextraction chambers were placed on each sonicated site of the humanvolunteer. Thin circular foam chamber 602 was constructed using foam MED5636 Avery Dennison ( 7/16″ ID× 11/8″ OD). Foam chambers 602 wereattached concentrically to the sonicated biological membrane sites usingdouble-sided adhesive (Adhesive Arcade 8570, 7/16″ ID×⅞″ OD) attached toone side of element 602. The other side of foam chamber 602 was attachedconcentrically to double-sided adhesive 604 (Adhesive Arcade 8570, 7/16″ID× 7/8″ OD). Thin transparent lid 606 was made of 3M Polyester 1012 (11/8″× 11/8″). Double-sided adhesive 604 permitted thin transparent lid606 to be attached to foam chamber 602 after placement of liquid intothe inner diameter of foam chamber 602 when attached to biologicalmembrane. Thin transparent lid 606 acted as a lid to prevent liquid fromleaking out of the extraction chamber, and to allow the extractionchambers to be wearable for an extended period of time.

Each extraction chamber was alternately filled with 100 μl of extractionsolution for 15 min and 100 μl hydration solution for 10-40 min.Extraction solution was PBS; on two sites the PBS contained additionalNaCl to bring the total concentration of NaCl to 1 M. Hydration solutionwas PBS for all sites.

Solutions were collected and analyzed for glucose concentration usinghigh-pressure liquid chromatography. The results of the HPLCconcentration were normalized for the injection amount and the totalsolution volume, and were reported as glucose flux (Q_(g)), the mass ofglucose that crossed the sonicated site per unit time per unit area.Body fluid glucose concentrations (C_(bg)) were obtained by testingcapillary blood obtained from a lanced finger in a Bayer GlucometerElite meter. It was hypothesized that Q_(g) would be linearlyproportional to C_(bg). FIG. 7 shows a graph of Q_(g) versus C_(bg).Unexpectedly, Q_(g) from the sonicated sites exposed to 1 M NaClcorrelated to C_(bg) much more strongly than Q_(g) from the sonicatedsites exposed to 0.15 M NaCl.

According to another aspect of the present invention, an apparatus andmethod for regulating the degree of skin permeabilization through afeedback system is provided. This apparatus and method may be similar towhat has been described above, with the addition of further regulationof the degree of skin permeabilization. Feedback control as a method ofmonitoring the degree of skin permeability is described in more detailin U.S. application Ser. No. 09/868,442, entitled “Methods and Apparatusfor Enhancement of Transdermal Transport,” which is hereby incorporatedby reference in its entirety. In this embodiment, the application of theskin permeabilizing device is terminated when desired values ofparameters describing skin conductance are achieved. As the discussionproceeds with regard to FIG. 8, it should be noted that the descriptionsabove may be relevant to this description.

Referring to FIG. 8, a flowchart of the method is provided. In step 802,a first, or source, electrode is coupled in electrical contact with afirst area of skin where permeabilization is required. The sourceelectrode does not have to make direct contact with the skin. Rather, itmay be electrically coupled to the skin through the medium that is beingused to transmit ultrasound. In one embodiment, where anultrasound-producing device is used as the skin permeabilizing device,the ultrasonic transducer and horn that will be used to apply theultrasound doubles as the source electrode through which electricalparameters of the first area of skin may be measured and is coupled tothe skin through a saline solution used as an ultrasound medium. Inanother embodiment, a separate electrode is affixed to the first area ofskin and is used as the source electrode. In still another embodiment,the housing of the device used to apply ultrasound to the first area ofskin is used as the source electrode. The source electrode can be madeof any suitable conducting material including, for example, metals andconducting polymers.

Next, in step 804, a second, or counter, electrode is coupled inelectrical contact with a second area of skin at another chosenlocation. This second area of skin can be adjacent to the first area ofskin, or it can be distant from the first area of skin. The counterelectrode can be made of any suitable conducting material including, forexample, metals and conducting polymers.

When the two electrodes are properly positioned, in step 806, an initialconductivity between the two electrodes is measured. This may beaccomplished by applying an electrical signal to the patch of skinthrough the electrodes. In one embodiment, the electrical signalsupplied may have sufficient intensity so that the electrical parameterof the skin can be measured, but have a suitably low intensity so thatthe electrical signal does not cause permanent damage to the skin, orany significant electrophoresis effect for the substance beingdelivered. In one embodiment, a 10 Hz AC source is used to create avoltage differential between the source electrode and the counterelectrode. The voltage supplied should not exceed 500 mV, and preferablynot exceed 100 mV, or there will be a risk of damaging the skin. Inanother embodiment, an AC current source is used. The current source mayalso be suitably limited. The initial conductivity measurement is madeafter the source has been applied using appropriate circuitry. In oneembodiment, a resistive sensor is used to measure the impedance of thepatch of skin at 10 Hz. In another embodiment, a 1 kHz source is used.Sources of other frequencies are also possible.

In step 808, a skin permeabilizing device is applied to the skin at thefirst site. Any suitable device that increases the permeability of theskin may be used. In one embodiment, ultrasound is applied to the skinat the first site. According to one embodiment, ultrasound having afrequency of 20 kHz and an intensity of about 10 W/cm² is used toenhance the permeability of the patch of skin to be used for transdermaltransport.

In step 810, the conductivity between the two sites is measured. Theconductivity may be measured periodically, or it may be measuredcontinuously. The monitoring measurements are made using the sameelectrode set up that was used to make the initial conductivitymeasurement.

In step 812, mathematical analysis and/or signal processing may beperformed on the time-variance of skin conductance data. Experimentswere performed on human volunteers according to the procedure above,with ultrasound used as the method of permeabilization. Ultrasound wasapplied until the subjects reported pain. Skin conductivity was measuredonce every second during ultrasound exposure. After plotting theconductance data, the graph resembled a sigmoidal curve. The conductancedata was in a general sigmoidal curve equation:C=C _(i)+(C _(f) −C _(i))/(1+e ^(S(t-t*)))where:

-   -   C is current;    -   C_(i) is current at t=0;    -   C_(f) is the final current;    -   S is a sensitivity constant;    -   t* is the exposure time required to achieve an inflection point;        and    -   t is the time of exposure.

Referring again to FIG. 8, in step 814, the parameters describing thekinetics of skin conductance changes are calculated. These parametersinclude, inter alia, skin impedance, the variation of skin impedancewith time, final skin impedance, skin impedance at inflection time,final current, exposure time to achieve the inflection time, etc.

In step 816, the skin permeabilizing device applied in step 808 isterminated when desired values of the parameters describing skinconductance are achieved. For instance, when the skin conductanceincreases to a certain value, the permeabilizing device may bedeactivated. Alternatively, when the rate of change in the value of skinconductance is a maximum, the permeabilizing device may be deactivated.Additional details of the method for regulating the degree of skinpermeabilization are disclosed in the aforementioned U.S. applicationSer. No. 09/868,442.

A preferred embodiment of a continuous transdermal glucose monitoringsystem and method is described in connection with FIGS. 9-11. Asdiscussed above, the term “body fluid” may include blood, interstitialfluid, lymph, and/or analyte. Body fluids include, for example, bothcomplete fluids as well as molecular and/or ionic components thereof.Preferred embodiments of the invention may involve extraction andmeasurement of just the analyte.

FIG. 9 is a drawing of a continuous glucose monitoring system accordingto an exemplary embodiment of the invention. In this embodiment, thesystem includes a sensor assembly generally including a sensor body 901and a backing plate 902 as well as other components as described herein.The sensor body may include electrodes, as shown in FIG. 10, on itssurface for electrochemical detection of analytes or reaction productsthat are indicative of analytes. A thermal transducer 903, which may behoused in a housing with a shape that corresponds to that of the sensorbody 901, is located between the sensor body 901 and the backing plate902. Electrochemical sensors, such as hydrogen peroxide sensors, can besensitive to temperature fluctuation. The thermal transducer 903 may beused to normalize and report only those changes attributed to a changein analyte or analyte indicator. An adhesive disc 904 may be attached tothe side of the sensor body 901 that faces the thermal transducer 903.An adhesive ring 905 may be attached to the side of the sensor body 901that is opposite the adhesive disc 904. The cut-out center portion ofthe adhesive ring 905 preferably exposes some or all of the sensorcomponents on the sensor body 901. The adhesive ring 905 and adhesivedisc 904 may have a shape that corresponds to that of the sensor body asshown in FIG. 9. A hydrogel disc 906 may be positioned within thecut-out center portion of the adhesive ring 905 adjacent a surface ofthe sensor body 901. During operation, the sensor assembly may bepositioned adjacent a permeable region 907 of a user's skin as shown bythe dashed line in FIG. 9. The sensor assembly may be attached to apotentiostat recorder 908, which may include a printed circuit board911, by way of a flexible connecting cable 909. The connecting cable 909preferably attaches to the potentiostat recorder 908 using a connector910 that facilitates removal and attachment of the sensor assembly.

The system shown in FIG. 9 can be used to carry out continuousmonitoring of an analyte such as glucose as follows. First, a region ofskin on the user is made permeable using, for example, sonication asdescribed above. The sensor assembly, such as that shown in FIG. 9, isthen attached to the permeable region 907 of skin so that the hydrogeldisc 906 is in fluid communication with the permeable skin. An analytemay be extracted through the permeable region 907 of the user's skin sothat it is in contact with the hydrogel disc 906 of the sensor assembly.For example, an analyte such as glucose may be transported by diffusioninto the hydrogel disc 906 where it can contact glucose oxidase. Theglucose can then react with glucose oxidase present in the hydrogel disc906 to form gluconic acid and hydrogen peroxide. Next, the hydrogenperoxide is transported to the surface of the electrode in the sensorbody 901 where it is electrochemically oxidized. The current produced inthis oxidation is indicative of the rate of hydrogen peroxide beingproduced in the hydrogel, which is related to the amount of glucose fluxthrough the skin (the rate of glucose flow through a fixed area of theskin ). The glucose flux through the skin is proportional to theconcentration of glucose in the blood of the user. The signal from thesensor assembly can thus be utilized to continuously monitor the bloodglucose concentration of a user by displaying blood glucoseconcentration on the potentiostat 908 in a continuous, real-time manner.

Detailed views of a preferred embodiment of the sensor body 901 areshown in FIG. 10. The sensor body 901 includes a body layer 1007 uponwhich leads 1004, 1005, and 1006 are patterned. The leads may be formed,for example, by coating metal over the body layer 1007 in the desiredlocations. A working electrode 1001, is typically located at the centerof the sensor body 901. The working electrode 1001 may comprise pureplatinum, platinized carbon, glassy carbon, carbon nanotube, mezoporousplatinum, platinum black, paladium, gold, or platinum-iridium, forexample. The working electrode 1001 may be patterned over lead 1006 sothat it is in electrical contact with the lead 1006. A counter electrode1002, preferably comprising carbon, may be positioned about theperiphery of a portion of the working electrode 1001, as shown in FIG.10. The counter electrode 1002 may be patterned over lead 1005 so thatit is in electrical contact with the lead 1005. A reference electrode1003, preferably comprising Ag/AgCl, may be positioned about theperiphery of another portion of the working electrode 1001 as shown inFIG. 10. The electrodes 1001, 1002, and 1003 can be formed to roughlytrack the layout of the electrical leads 1006, 1005, 1004, respectively,that are patterned in the sensing area of the device. The electrodes1001, 1002, and 1003 may be screen printed over the electrical leads1006, 1005, 1004, respectively. The leads can be pattered, using screenprinting or other methods known in the art, onto the sensor body 901 ina manner that permits electrical connection to external devices orcomponents. For example, the leads may form a 3X connector pin leadincluding leads 1004, 1005, and 1006 at the terminus of an extendedregion of the sensor body as shown in FIG. 10. A standard connector maythen be used to connect the sensor electrodes to external devices orcomponents.

The electrochemical sensor utilizes the working electrode 1001, thecounter electrode 1002, and the reference electrode 1003 to measure therate hydrogen peroxide or glucose is being generated in the hydrogel.The electrochemical sensor is preferably operated in potentiostat modeduring continuous glucose monitoring. In potentiostat mode, theelectrical potential between the working and reference electrodes of athree-electrode cell are maintained at a preset value. The currentbetween the working electrode and the counter electrode is measured. Thesensor is maintained in this mode as long as the needed cell voltage andcurrent do not exceed the current and voltage limits of thepotentiostat. In the potentiostat mode of operation, the potentialbetween the working and reference electrode may be selected to achieveselective electrochemical measurement of a particular analyte or analyteindicator. Other operational modes can be used to investigate thekinetics and mechanism of the electrode reaction occurring on theworking electrode surface, or in electroanalytical applications. Forinstance, according to an electrochemical cell mode of operation, acurrent may flow between the working and counter electrodes while thepotential of the working electrode is measured against the referenceelectrode. It will be appreciated by those skilled in the art that themode of operation of the electrochemical sensor may be selecteddepending on the application.

The sensor assembly described generally in relation to FIG. 9 is show inexpanded detail from another angle in FIG. 11. The sensor body 901,which is covered on each side by adhesive disc 904 and adhesive ring905, is shown in relation to the backing plate 902. The hydrogel disc906 may be positioned in such a manner that it will face toward the userafter folding over onto the backing plate 902 as shown in FIG. 9. Thesensor body may be connected to the backing plate 902 using standardconnectors such as a SLIM/RCPT connector 1301 with a latch that mateswith a corresponding connector interface that is mounted onto thebacking plate 902.

The sensor assembly shown in FIGS. 9-11 may be incorporated into any oneof a number of detection devices. For instance, this sensor assembly maybe incorporated into the receiver of FIG. 4 to provide for discreteand/or continuous glucose monitoring. Additionally, the sensor assemblymay be connected to a display or computing device through a wirelessconnection or any other means for electrical connection in addition tothe cable 909.

Continuous glucose monitoring as described herein can be achievedwithout accumulation of a certain volume of body fluid in a reservoirbefore measuring the concentration of the withdrawn fluid. Continuousglucose monitoring is capable of measuring the blood concentration ofglucose without relying on accumulation of body fluids in the sensordevice. In continuous glucose monitoring, for instance, one may preferto minimize accumulation of both glucose and hydrogen peroxide in thehydrogel so that the current measured by the electrochemical sensor isreflective of the glucose flux through the permeable region of skin inreal-time. This advantageously permits continuous real-time transdermalglucose monitoring.

According to another aspect of the invention, a step of skin hydrationmay be employed prior to or concurrently with increasing the porosity ofthe skin (e.g. by applying ultrasound) to improve the continuoustransdermal analyte monitoring. Skin hydration prior to or concurrentlywith increasing the porosity, and prior to attaching the sensor mayimprove sensor performance by establishing or stabilizing liquidpathways between the skin and the sensor, improving the moisture balanceover the sensor-skin interface, and/or continuing to maintain amplewater to the hydrogel to maintain enzyme activity. The skin hydrationprocedure can be performed, for example, by applying a hydrating agentto the target skin site. The hydrating agent may be applied incombination with a delipidation or cleansing agent. Where both hydratingand cleansing agents are utilized, they may be applied in a singleapplication using a single solution. Alternatively, the cleansing agentand the hydrating agent can be applied using successive application oftwo different solutions. In one aspect, one or both solutions areapplied using a pad applicator. In another aspect, the solution can beheld in contact with the skin by positioning it in the bellows of asonication device or another device that might function to hold a liquidin contact with skin.

In one embodiment, a glycerin/water prep pad solution may be preparedfor skin hydration. The following batch formulation can be used toprepare the glycerin/water prep pad solution. 300.00 grams of glycerin99% USP is added to the first container. 2.70 grams of Nipagin M(methylparaben), 0.45 grams of Nipasol M (propylparaben), and 30.00grams of benzyl alcohol NF are dissloved in a second container and thenadded to the first container. The glycerin and benzyl alcohol solutionsare then mixed in the first container until the solution clears. 1133.85grams of deionized water is then added to the solution in the firstcontainer and mixed until homogeneous. 1.50 grams of Potassium SorbateNF is added to the solution in the first container and mixed untilhomogenious. 1.50 grams of Glydant 2000 is then added to the solution inthe first container and mixed until homogenious. Lastly, 30.00 grams ofdeionized water is added to the solution in the first container andmixed until homogeneous.

In one embodiment, a 1 3/16″ prep pad is utilized. Preferably the preppads are composed of 70% polypropylene/30% cellulose. In one embodiment,the prep pad has a width that ranges from 1 1/16″ to 1 5/16″. In oneembodiment, the thickness of the prep pad is 21-29 mils. In anotherembodiment, the thickness of the prep pad is 26-34 mils. In oneembodiment the prep pad has a basis weight of 1.43-1.87 g/yd usingATM#102. In another embodiment, the prep pad has a basis weight of1.72-2.24 g/yd using ATM#102. Preferably, the prep pad is utilized witha prep pad solution, such as the prep pad solution above, to hydrate abiological membrane before increasing its porosity.

According to another aspect of the invention, the working electrode 1001of FIG. 10 may include a surface layer of pure platinum. The pureplatinum working electrode 1001 may be screen printed or otherwisecoated onto the surface of a lead 1006. Using pure platinum as theworking electrode can enhance sensitivity and increase the rate ofconversion of hydrogen peroxide. This can provide advantages forcontinuous transdermal glucose monitoring as the conversion of hydrogenperoxide is preferably fast to prevent its accumulation, which may causepositive sensor drift and/or enzyme deactivation. In transdermal glucosesensing applications, pure platinum can offer advantages overtraditional platinized carbon materials.

One advantage that pure platinum can offer relative to platinized carbonis an enhanced sensitivity to glucose concentration. FIG. 13 shows theglucose sensitivity of both pure platinum and platinized carbon. Asshown by this comparison, the glucose sensitivity of pure platinum isabout 2.9 times that of platinized carbon. The glucose sample size usedto generate the data of FIG. 13 was 2 microliters.

Another advantage that pure platinum can offer relative to platinizedcarbon is enhanced sensitivity to hydrogen peroxide. FIG. 14 shows thehydrogen peroxide sensitivity of both pure platinum and platinizedcarbon. Specifically, FIG. 14 shows the current-time profiles of aglucose sensor responding to the addition of hydrogen peroxide(sometimes referred to as a hydrogen peroxide “challenge”) usingplatinum and platinized carbon as the working electrode. As shown bythis comparison, the hydrogen peroxide sensitivity of pure platinum isabout 5 times that of platinized carbon.

Another advantage that pure platinum can offer relative to platinizedcarbon is a higher success rate for glucose monitoring. The percentagesuccess rate for glucose monitoring using pure platinum was 83% versus60% for platinized carbon (correlation coefficient R²>=0.5 as thepassing criteria). R refers to the correlation between conventionalwhole blood glucose measurements and measurements of blood glucose usingthe system of FIG. 9. R is calculated by comparing the continuous datafrom the system of FIG. 9 with discrete whole blood measurements (takenevery 20 minutes). A linear regression analysis is run on the two datasets to generate an R value. The correlation between sensor signal andblood glucose levels using pure platinum was R²=0.87 versus R²=0.71 forplatinized carbon.

According to another aspect of the invention, a protective interferencefilter can be provided to reduce or even eliminate interference effectsfrom unwanted electrochemical oxidation and/or biofouling. One form ofinterference, for example, involves the production of unwanted anodicsignal by electrochemical oxidation of ascorbic acid, uric acid, and/oracetaminophen, which can all be oxidized electrochemically at voltagelevels applied in glucose monitoring. Another form of interference caninvolve biofouling, which can occur when biological species deposit on asensor surface thereby limiting the sensor's free access to analyte ordeactivating its functionality by reacting with the electrode. It isgenerally advantageous to reduce or eliminate the effects of interferingspecies through the use of an interference filter since many of thesespecies may be present in body fluids during glucose monitoring.

According to an exemplary embodiment of the invention, the interferencefilter comprises a Nafion film coated onto one or more surfaces of thesensor assembly. Other interference filter materials such as(3-mercaptopropyl)trimethylsilane, cellulose acetate, electropolymerizedfilms such as 1,8-diaminonapthaline and phenylenediamine, PTFE or otherhydrophobic, Nylon or other hydrophylic membranes may be used. Nafion isa biocompatible anionic fluoropolymer that can be coated on sensorsurfaces as a protective layer against physiological interferents andbiofouling based on hydrophobicity, charge selection, and sizeexclusion, for example. Nafion is available from Aldrich Chemical ofMilwaukee, Wis. A Nafion film may be coated directly on the surface ofat least the working electrode 1001 of the sensor body 901.Alternatively, a Nafion film may be coated on an outer surface of thesensor assembly such as the hydrogel layer 906. In general, one or moreinterference filter layers may be provided between the working electrodesurface and any other layer or on the outermost surface of the sensorassembly that contacts the user's skin during operation.

A Nafion layer can be conveniently coated on a sensor surface using amicropipette, for example, or by dip-coating the sensor in aqueous ororganic Nafion solution followed by air drying for several hours beforeuse. FIG. 15 shows the effect of a Nafion coating on the sensor responseto glucose relative to the interferents acetaminophen and uric acid. Theplot shows the hydrodynamic sensor response to 0.294 mM of hydrogenperoxide (HP) over acetominophen (AM) and uric acid (UA) in phosphatebuffered saline with 0.5 V of applied voltage. The amperometric currentproduced by acetaminophen and uric acid is greatly reduced for a sensorcoated with Nafion relative to an uncoated sensor. Thus, Nafion cansignificantly improve the analyte/interferent signal ratio.

In various embodiments of the invention described herein, hydrogels canbe used as part of the analyte monitoring system. Hydrogels constitutean important class of biomaterials utilized for medical andbiotechnological applications such as in contact lenses, biosensors,linings for artificial implants and drug delivery devices. FIGS. 9 and11 show a preferred hydrogel disc 906 in relation to the sensorassembly. The hydrogel disc 906 may be located over the sensor body 901within the cutout center portion of the adhesive ring 905 of the sensorassembly. The continuous transdermal analyte monitoring system mayutilize one or more of the preferred hydrogel materials described below.Classes of hydrogel materials that may be used in exemplary embodimentsof the invention include: agarose based hydrogels, polyethylene glycoldiacrylate (PEG-DA) based hydrogels, and vinyl acetate based hydrogels,for example. Following a general description of these gels are examplesdetailing the procedures used to produce and/or characterize the varioushydrogels.

Agarose based hydrogels can offer advantages for continuous transdermalanalyte monitoring. For instance, agarose can offer one or more of thefollowing features: good response to glucose and hydrogen peroxide dueto its high water content, high enzyme loading, good bio-compatibility,and excellent permeation and diffusion properties. In addition, agarosehydrogels may offer cleanliness, low cost, and/or ease of preparation.

An agarose gel may be formed, for example, from 1-20% agarose in buffersolution containing 0-1 M sodium or potassium phosphate, 0-1 M sodiumchloride, 0-1 M potassium chloride, 0-2 M lactic acid, surfactant suchas 0-1 M Triton X-100, Tween 80 or sodium lauryl sulfate, and any otherbiocompatible components. Loading of glucose oxidase in agarose hydrogelcan be up to 0-20% (by weight), for example, by soaking the solidhydrogel in concentrated glucose oxidase solution, or alternatively bymixing concentrated glucose oxidase powder or solution with agarosesolution during its melting stage (15-65° C.), followed by cooling andgelling at lower temperature (40° C. or lower).

PEG based hydrogels can offer several advantages for continuoustransdermal analyte monitoring. Structurally, PEG is highly hydrophilicand presents a high degree of solvation in aqueous solvents. Thepreferential solvation of PEG molecules can effectively exclude proteinsfrom the PEG chain volume, thereby protecting the surface frombio-fouling by proteins. An advantage that can be provided by chemicallycrosslinked PEG-based hydrogels is that their physical and chemicalproperties can be modulated by varying the molecular weight of the PEGchains and varying the initiator concentration. For example, increasingthe molecular weight of the polyethylene oxide backbone increases thenetwork mesh size. The release of a bioactive molecule such as an enzymecan be controlled by control of the network density. Therefore, ahydrogel comprised of PEGs of molecular weight 8000 daltons would have ahigher rate of release of an entrapped drug than a hydrogel comprised ofPEGs of molecular weight 3.3K. Furthermore, ionic moieties can beincorporated into the hydrogels to impart added functionalities such asbioadhesiveness, etc. For example, hyaluronic acid or polyacrylic acidcan be added to the PEG macromer prior to crosslinking to createbioadhesive hydrogels. In another example, an ionic character can beimparted to the crosslinked hydrogels to provide molecular interactionwith entrapped drugs to slow down rates of release of drug from thematrix.

PEG-hydrogels used in biosensors can provide one or more of thefollowing features: (a) a biocompatible, non-biofouling surfaceappropriate for long-term exposure to biological fluids withoutcompromise of sensor function, (b) a reservoir for glucose oxidase, (c)a matrix that can be incorporated with ionic moieties to enhanceentrapment of glucose oxidase, (d) a matrix that can be modulated interms of its physical and chemical properties (network density,swelling) by varying the molecular weight of the backbone and (e) amatrix that can be rendered bioadhesive by addition of ionic excipientssuch as chitosan gluconate, polyacrylic acid, poly(amidoamine),poly(ethyleneimine) and hyaluronic acid.

Vinyl acetate based hydrogels, such as n-vinylpyrolidone/vinyl acetatecopolymer, can exhibit features such as transparency, tackiness,non-toxicity, flexibility, and/or hydrophobicity. Vinyl acetate basedhydrogels typically have a good ability to retain moisture and entrapenzymes such as glucose oxidase, biocompatibility, and tackiness to skinto improve skin-sensor coupling. A glucose flux sensor usingn-vinylpyrolidone/vinyl acetate copolymer as the hydrogel material showsgood performance in tracking the plasma glucose levels of a patient withdiabetes during a glucose clamping study.

The following examples set forth exemplary hydrogels that can be usedwith transdermal analyte monitoring according to embodiments of thepresent invention.

EXAMPLE 2

Vinyl acetate based hydrogels for use with glucose monitoring can beprepared as follows. A 1:1 mixture of n-vinylpyrolidone and vinylacetate can be polymerized by ultraviolet radiation using 0-0.5%Irgacure as the photoinitiator. A non-woven plastic scrim (such asDelstar product# RB0707-50P) is used to provide mechanic support. Thehydrogel's equilibrium water content is 20-95% with its aqueouscomposition containing 0-1 M sodium or potassium phosphate, 0-1 M sodiumchloride, 0-1 M potassium chloride, 0-2 M lactic acid, surfactant suchas 0-1 M Triton X-100, Tween 80 or sodium lauryl sulfate, and any otherbiocompatible components. Glucose oxidase can be loaded by soaking thesolid hydrogel layer in concentrated glucose oxidase solution for aperiod of time.

A particular example of a vinyl acetate based hydrogel was made with thefollowing constituents: 15% n-vinylpyrolidone, 15% vinyl acetate, 0.05%Irgacure, 0.05 M potassium phosphate, 0.30 M sodium chloride, 0.025 Mpotassium chloride, 0.5 M lactic acid, 0.1% Triton X-100, 0.5% GOx, andthe remaining composition is water, approximately 65%

The continuous transdermal analyte monitoring system according to anexemplary embodiment of the present invention was used to reliablypredict hypoglycemia (blood glucose<70 mg/dl) with 96% specificity and77% sensitivity using a vinyl acetate hydrogel. In a study, thirty sixglucose flux biosensors (3 per patient) were placed on the skin oftwelve adults with either Type 1 or Type 2 diabetes. Patient data forparticipants in the study are shown in FIG. 24. Blood glucosemeasurements were collected over an eight hour period. Thesemeasurements included collecting current versus time data from thepatients using a continuous transdermal analyte monitoring system asdescribed herein. The blood glucose of each patient was rapidlyincreased or decreased through the administration of insulin or glucoseintravenously in a controlled manner at a rate of change two timesgreater than that usually experienced by patients with diabetes.Specifically, the ranges tested were 35-372 mg/dl blood glucose, with arate of glucose concentration decrease of 21 mg/(dl*min) and rate ofglucose concentration increase of 11 mg/(dl*min). As a control, bloodglucose measurements were collected from an intravenous catheter. Atotal of 2039 sensor-blood glucose data pairs from 29 data sets weregenerated. Five of the data sets had significant noise as shown in FIG.25. The typical data set, however, kept noise below an excessive levelas shown, for example, in FIG. 26. The data sets were analyzed with bothan individually optimized algorithm and an independent algorithm, andthe results are shown in FIGS. 27 and 28, respectively. The individuallyoptimized algorithm used each data set's optimal lag time and baselinefor data analysis. The independent algorithm was developed from aseparate glucose clamping study, from which a single lag time value anda single baseline value were found, then were used in the algorithm fordata analysis. As will be described below in connection with FIG. 17, anadditional algorithm can also be utilized to compensate for temperaturechange and sensor drift. Completed data sets from the glucose biosensorsshowed a 90 percent (R=0.9) correlation to blood glucose measurementsobtained via intravenous catheter over a period of 8 hours. Ninety sixpercent of the sensor-blood glucose pairs fell within the A+B regions inthe Clark Error Grid. Seventy seven percent (164 out of 212)hypoglycemic events (BG<70 mg/dL) were successfully predicted.Sonication treatment (using Sonoprep) averaged 15 seconds and theglucose sensor required only 89+/−6 minutes on average to break in. Nopain or irritation was reported during the sonication procedure.Accordingly, the glucose biosensor was able to reliably predicthypoglycemia (blood glucose<70 mg/dl) with 96% specificity and 77%sensitivity.

EXAMPLE 3

Agarose based hydrogels for use with glucose monitoring were prepared asfollows. 0.0116 g of sodium chloride, 0.015 g of potassium chloride,0.0348 g of dibasic potassium phosphate and 0.002 g of Triton X-100 weredissolved in 10 mL of water. The pH of the solution was adjusted to 7.0using 0.5 M hydrochloric acid with the aid of a pH meter. The solutionwas diluted with water to 20 mL. This was Solution A. 0.2 g of agarosepowder was mixed and dispersed in Solution A. Agarose was heated anddissolved until boiling in a water bath. This was Solution B. Solution Bwas allowed to cool down to 35° C. 0.01 g of glucose oxidase powder wascompletely mixed and dissolved in Solution B. This was Solution C.Solution C was cast and filled onto a warm, flat mold surface. The moldwas transferred to room temperature or lower to form gels.

FIG. 12 shows sensor signal response as a function of glucoseconcentration for two types of agarose hydrogels relative to apolyethylene oxide polymer, and a n-vinyl pyrolidone/vinyl acetatecopolymer. It can be seen from FIG. 12 that agarose offers improvedsignal response relative to polyethylene oxide polymer and n-vinylpyrolidone/vinyl acetate copolymer.

EXAMPLE 4

Agarose based hydrogels for use with glucose monitoring can also beprepared as follows. Mix and disperse 0.2 g of agarose powder in water.Heat and dissolve agarose until boiling in a water bath. Cast and fillthe solution onto a warm, flat mold surface. Transfer the mold to roomtemperature or lower to form gels. Dissolve 0.01 g of glucose oxidasepowder in Solution A to form Solution D. Soak the gel in Solution Dovernight or longer to ensure sufficient loading of glucose oxidase inthe gel.

EXAMPLE 5

PEG-diacrylate (PEGDA) hydrogels utilized in glucose monitoring wereprepared according to the following procedures.

10% weight/volume (“w/v”) solutions of (100 mg/ml) PEG2K-diacrylate,PEG3.4K-diacrylate and PEG8K-diacrylate (SunBio, Korea) were prepared in0.01M phosphate buffered saline (PBS), pH 7.4 (ultrapure, SpectrumChemicals, Gardena, Calif.). The solutions all contained Irgacure 2959(Ciba Specialty Chemicals, Tarrytown, N.Y.) as the photoinitiator.Irgacure concentrations were varied to determine the effect ofphotoinitiator concentration on gel strength. Similarly, the polymermolecular weights were varied (2K, 3.4K, 8K) to determine the effect ofmolecular weight on the strength of the gelled network. As used herein,the notation “PEG2K” refers to PEG having a molecular weight of 2,000,etc.

100 mg of dry polymer was weighed into a scintillation vial. 900 μl ofphosphate buffered saline (PBS) containing 500 ppm of Irgacure 2959 wasadded to the vial and the final weight of the solution was recorded. Thevial was screw-capped and the vial swirled gently to dissolve the PEGDA.The gel solution was stored in the drawer (in the dark) for 5 minutes toensure homogeneity. 900 μl of the gel solution was placed between twoglass plates (250μ spacers) and clamped. The glass assembly containingthe polymer solution was placed under a UV Blak-Ray lamp, at anintensity of 15-20 mW/cm² and photo-crosslinked between 5-30 minutes.The gel was removed carefully from the glass and weighed beforetransferring to 10 ml of PBS in a plastic petri dish. After removal fromthe glass plates, the hydrogels were placed in approximately 10 ml ofPBS. The hydrogels were then qualitatively assessed for bulk gelproperties such as brittleness, gel strength and photo-yellowing as afunction of molecular weight and initiator concentration.

The following procedure was used to measure the equilibrium hydration ofthe gels. The gels were weighed after curing was complete. The initialweight of the gel was obtained, post wiping gently with a Kim-wipe. 10ml of PBS was added to the petri dish containing the gels. The petridishes were placed on an orbital shaker. The buffer was replaced atpre-determined time intervals. The retrieved buffer solutions were savedto analyze for residual Irgacure. At each time interval, the gel waswiped dry with a Kim Wipe and weighed. The percent swelling (%hydration) was calculated by the change in total weight as compared tothe initial weight of the gel.

By qualitative assessment, the gels varied in gel strength in thefollowing order (strongest gel to weakest gel): PEG8K>PEG3.4K>PEG2K. Gelstrength was ascertained by degree of pliability, ease of handling, andbrittleness. Gel strength was also noted to vary with concentration ofthe photoinitiator, with higher concentrations yielding hydrogels thatwere hard and brittle. Photoyellowing from Irgacure photoinitiation wasnoted in hydrogels in the following order (most photoyellowing to leastphotoyellowing): 5000 ppm>2500 ppm>1500 ppm>500 ppm. The photoinitiatorconcentration of 500 ppm and a PEGDA molecular weight of 8K resulted inthe highest gel strength.

The following procedures were performed to incorporate glucose oxidase(GOx) into the gels. First, the gels were tested for residual Irgacure2959. Next a glucose oxidase solution was prepared. The glucose oxidasewas then loaded into the PEGDA hydrogels. The glucose oxidaseconcentration in the gels was measured. Lastly, the bioactivity of thegels was measured. The following describes these steps in detail.

The hydrogels were washed twice with buffer until there was nodetectable residual Irgacure extracted from the hydrogels. The washsolutions were scanned on the UV-Vis from 200-400 nm, for the presenceof Irgacure 2959. Non-detectable levels of Irgacure were determined tobe an absorbance at 280 nm<0.010, equivalent to 0.13 ppm, as compared toa 25 ppm Irgacure solution that had an absorbance of 1.8 at 280 nm.

An LPT buffer solution was prepared by mixing 5% w/v glucose oxidase inPBS solution with 0.25 M lactic acid and 0.05% Triton X-100. This wasaccomplished by adding 0.5 grams of GOx to a total volume of 10 ml of astock solution comprised of 0.25 M lactic acid and 0.05% Triton X-100dissolved in PBS. The solution was kept at 4° C.

PEGDA hydrogels comprised of varying PEG molecular weights (2K, 3.4K,8K) were soaked in the glucose oxidase solution. The gels were soakedfor overnight or longer at 4° C., but no more than seven days.

Glucose oxidase concentrations were measured by the Bradford Assay, amethod commonly used to determine concentrations of solubilized protein.The method involves addition of an acidic blue dye (Coomassie BrilliantBlue G-250) to a protein solution. The dye binds primarily to basic andaromatic amino acid residues, especially arginine, with the absorptionmaximum shifting from 465 nm to 595 nm with complete dye-proteinbinding. The molar extinction coefficient of the dye-protein complex hasbeen determined to be constant over a 10-fold concentration range;therefore, Beer-Lambert's Law can be utilized to accurately determineconcentrations of protein. A standard curve of glucose oxidase solutionsat concentrations 0.125%, 0.25%, 0.375%, 0.5% and 2.5% w/v was obtainedby UV-Vis Spectroscopy at 595 nm after treatment of the standardsolutions and the gel fragments with standard Bradford protein assay dyeprocedure. See Bradford Assay, BioRad Laboratories Brochure. A linearcorrelation of 0.999 was obtained for the standard curve. GOxincorporation in the hydrogels was determined in the following method:(a) a piece of gel was soaked in 4 ml LPT solution containing 1 ml ofprotein assay dye concentrate, (b) A piece of GOx-soaked then dyed(Coomassie dye) hydrogel was sandwiched between two glass cuvettes, (c)a non-GOx soaked and dyed hydrogel was used in the reference cell, (d)The gels were scanned from 400-800 nm and (e) the concentration of GOxincorporated in the hydrogels were calculated from Beer Lambert's Law:A=εbc, where A=absorbance, ε=molar extinction coefficient, b=path lengthand c=concentration of the analyte. Concentrations of glucose oxidaseincorporated in 2K, 3.4K and 8K molecular weight PEG hydrogels weredetermined. FIG. 18(a) is a UV-Vis spectrum of a standard glucoseoxidase solution. FIG. 18(b) is an UV-Vis spectrum of Coomassie-boundglucose oxidase. The concentration in the gels is approximately 0.6%.

Electrochemical sensors were used to test the enzymatic activity of thehydrogel-incorporated GOx. Prior to the placement on sensor, the PEGDAhydrogels are cut to the diameter of the sensor surface and rinsedbriefly in LPT to remove surface residual GOx. Solutions of glucose(0.25 and 0.50 mg/dl) in PBS were used as the standard test solutionsand solutions of hydrogen peroxide (20 and 55 μM) in PBS were used asthe positive controls. Hydrogen peroxide, the reaction product ofglucose and GOx, produced amperometric current, which was recorded by apotentiostat connecting to the sensor. Therefore, positive sensor signalin response to a glucose challenge (addition of glucose) indicates thatthe incorporated enzyme was bioactive, while a positive sensor signal inresponse to a hydrogen peroxide challenge (addition of hydrogenperoxide) indicates that the eletrochemical sensor is functioning. PEGDAhydrogels with incorporated GOx were tested for peak signal strength andbaseline stability. These tests demonstrate that all hydrogels (2K,3.4K, 8K) contain bioactive GOx, and that 2K and 3.4K are advantageousfor signal strength and baseline stability (See FIGS. 19-20). FIG. 19shows the signal response to glucose of glucose oxidase loaded PEG gelsof varying molecular weight. FIG. 19 demonstrates that the PEG gelscontain bioactive GOx and that 2K and 3.4K molecular weight PEGhydrogels are advantageous for signal strength and baseline stability.FIG. 20 shows signal response to glucose of PEG3.4K-diacrylate hydrogelloaded with varying concentrations of GOx in the gel as well as for GOximmobilized on the sensor surface. The label “n” in FIGS. 19-20corresponds to the number of data sets that were taken with respect toeach condition tested. FIG. 21 shows the raw data of the potentiometricsignals elicited from PEGDA hydrogels with GOx incorporated in the gelformulation prior to photocrosslinking. The data from FIG. 21demonstrates that hydrogels with a thickness of 400 μm had significantnon-Gaussian peak shapes and tailing relative to gels at 200 μm, whichis indicative of slow diffusion of glucose and hydrogen peroxide throughthe hydrogel. FIG. 22 shows the change in signal between GOx-presoakedversus pre-incorporated, i.e., preloaded, hydrogels at different gelthickness and gel compositions (PEGDA-nVP, PEGDA). Among the variationsof gels tested were PEGDA hydrogels at varied thickness (200 μm, 400 μm)and PEGDA-nVP at 200 μm. The data from FIG. 22 demonstrates that the GOxincorporated in the hydrogels is bioactive. Baseline stability wasacceptable for all formulations and signals were not compromised.

The following describes ex vivo glucose testing on a patient withdiabetes using GOx loaded PEGDA hydrogel in a complete sensor assembly.The ultrasonic skin permeation procedure, sensing mechanism, sensorconfiguration and protocols for clinical trials are described in ChuangH, Taylor E, and Davison T., “Clinical Evaluation of a ContinuousMinimally Invasive Glucose Flux Sensor Placed Over UltrasonicallyPermeated Skin,” Diabetes Technology & Therapeutics, 6:21-30 (2004). Inthis clinical trial, PEGDA3.4K and pure platinum were used as thehydrogel and sensor materials, respectively.

Glucose sensor function using PEGDA hydrogel is shown in FIGS.23(a)-(b). FIG. 23(a) shows an example of sensor signal (nA) respondingcontinuously to changes of blood glucose (BG) levels in aglucose-clamping clinical study over a period of seven hours. Thecorresponding NA-BG correlation plot shown in FIG. 23 b has a Perason'scorrelation coefficient R=0.9476 (R²square=0.8979), revealing excellentsensor's function to monitor BG levels. Use of GOx loaded PEGDA hydrogelenables successful, continuous transdermal glucose monitoring.

EXAMPLE 6

PEG-diacrylate-n-vinyl pyrrolidone-GOx hydrogels (PEGDA-NVP) for usewith glucose monitoring were prepared according to the followingprocedures. PEGDA-NVP are slightly cationic, which provides ionicinteraction that retains GOx. Incorporating GOx within the hydrogelprior to crosslinking also contributes to physical entrapment of GOx inthe matrix. PEGDa-NVP hydrogels were prepared and characterizedaccording to the following procedure.

100 mg of dry polymer was weighed into a tared scintillation vial. 500μl PBS containing 1000 ppm of Irgacure 2959, 250 μl of 20% GOx in PBS,and 150 μl of 2% n-vinyl pyrrolidone (“n-VP”) was added to the vial andthe final weight of the solution was recorded. The vial was screw-cappedand the vial swirled gently to dissolve the PEGDA. The gel solution wasstored in the drawer (in the dark) for 5 minutes to ensure homogeneity.900 μl of the gel solution was placed between two glass plates (200μspacers) and clamped. The glass assembly containing the polymer solutionwas placed under an UV Blak-Ray lamp, at an intensity of 15-20 mW/cm²and cured for 5 minutes. The gel was removed carefully from the glassand weighed before transferring to 10 ml of LPT in a plastic petri dish.

The 200 micron hydrogels were transparent, easy to handle, pliable withconsiderable gel strength, as assessed qualitatively. Water content ofthe hydrogels were approximately 90%. The GOx was incorporated in thehydrogels prior to crosslinking, resulting in semi-interpenetratingnetworks. The hydrogels retained their yellow color (due to the GOx),post hydration. This indicated higher retention of the enzyme within thehydrogel.

Bioactivity of the incorporated enzyme was determined by potentiometry.This experiment demonstrated that glucose oxidase incorporated with PEGdiacrylate-n-vinyl pyrrolidone hydrogels is bioactive and chemicallycompatible with the hydrogel delivery system. Data in FIGS. 21-22demonstrate that GOx incorporated within the hydrogels are bioactive andfunctional.

EXAMPLE 7

PEG-diacrylate/Polyethyleneimine (PEGDA-PEI) hydrogels for use withglucose monitoring can be prepared according to the followingprocedures. PEGDA-PEI are cationic hydrogels. Polyethyleneimine(branched, or dendrimer, Sigma Chemicals) can be incorporated within PEGdiacrylate hydrogels to impart cationic character. A cationic hydrogelcan ionically interact with slightly anionic glucose oxidase to providea controlled release reservoir for the enzyme. A solution comprised of0.3-0.5% PEI, 10% PEGDA, 500 ppm Irgacure 2959 and 5% glucose oxidasecan be photocrosslinked with a BlakRay UV light, as described inprevious sections. Incorporation of the highly cationic PEI can providea high-binding substrate for GOx resulting in enhanced retention of theenzyme in the matrix. Furthermore, the highly cationic character of thehydrogels can provide the added functionality of bioadhesivity to theskin. Other cationic, bioadhesive macromolecules that can beincorporated into PEGDA hydrogels are chitosan, polyamidoamine,poly(n-vinyl pyrrolidone), etc.

According to another aspect of the invention, an error correction methodcan be utilized to correct for sensor drift in a measured blood glucosevalue as a function of time. FIG. 16 shows a Clark Error Grid withoutthe error correction method to correct for sensor drift. The data inFIG. 16 were taken from ten ex vivo tests on diabetic subjects in aclinical trial. The different data labels indicate data from differentpatients. FIG. 17 shows the Clark Error Grid after application of theerror correction method to correct sensor drift. The data in FIG. 17were taken from ten ex vivo tests on diabetic subjects in a clinicaltrial. The error correction method is described below.

The sensor signal, Y, as a function of time, t, is related to the sensorsensitivity, m, blood glucose value, X, and a constant offset value, b,according to the following linear relationship:Y=mX(t)+b

The above equation can be rearranged, and the blood glucose value can beconveniently predicted with a single point calibration protocol asfollows:X(t)=(Y−b)/m, and m=(Yc−b)/Xrc(t)

The value of sensor sensitivity, m, can be found from each ex vivo studyusing the sensor's current reading Yc and a standard reference bloodglucose value Xrc(t) at the sensor calibration time point. Whencomparing subsequent blood glucose value, X(t), with correspondingstandard reference blood glucose value Xr(t), it is found that a driftfactor D(t) can be computed at different points as follows:D(t)=Xr(t)/X(t)

By plotting D(t) vs. time, t, from a bulk number of successful ex vivostudies, a best fit for the D(t) vs. t plot was a third order polynomialfunction, which can be represented as follows:D(t)=c*t ³ +d*t ² +e*t+fwhere c, d, e, f are numerical coefficients calculated to provide thebest fit for the D(t) vs. t data to the above third order polynomial.The use of a third order polynomial is, however, exemplary and othermethods of representing the drift factor such as an algorithm fittingthe drift data to an exponential function, or utilizing a direct look-uptable method can also be utilized.

To predict a drift-corrected blood glucose value Xp(t) at time t, onecan simply multiply X(t) by D(t) as follows:Xp(t)=X(t)*D(t)=X(t)*(c*t ³ +d*t ² +e*t+f)

This equation represents an error correction method, and its utility maybe appreciated by a comparison of the Clark Error Grid where thealgorithm is not applied (FIG. 16) versus where it is applied (FIG. 17).The negative bias and wide scattering of data pairs in FIG. 16 iseffectively corrected, and as a result all data points fall in theclinically relevant A and B regions in the Clark Error Grid, as shown inFIG. 17. This error correction method may be applied to data generatedusing the continuous transdermal analyte monitoring system according anexemplary embodiment of the present invention.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All references cited herein,including all U.S. and foreign patents and patent applications, arespecifically and entirely hereby incorporated herein by reference. It isintended that the specification and examples be considered exemplaryonly, with the true scope and spirit of the invention indicated by thefollowing claims.

1. A transdermal analyte monitoring system comprising: a medium adaptedto interface with a biological membrane and to receive an analyte fromthe biological membrane; and an electrode assembly comprising aplurality of electrodes, wherein a surface region of at least one of theelectrode consists essentially of pure platinum; wherein the medium isadapted to react continuously with the analyte; and wherein anelectrical signal is detected by the electrode assembly, and theelectrical signal correlates to an analyte value.
 2. The transdermalanalyte monitoring system of claim 1, wherein the analyte value is theflux of the analyte through the biological membrane.
 3. The transdermalanalyte monitoring system of claim 1, wherein the analyte value is theconcentration of the analyte in a body fluid of a subject.
 4. Thetransdermal analyte monitoring system of claim 1, further comprising asensor body that supports the electrode assembly and the medium.
 5. Thetransdermal analyte monitoring system of claim 1, wherein the analytecomprises glucose.
 6. The transdermal analyte monitoring system of claim5, wherein the medium comprises a hydrogel.
 7. The transdermal analytemonitoring system of claim 6, wherein the medium further comprisesglucose oxidase.
 8. The transdermal analyte monitoring system of claim5, wherein the surface region consisting essentially of pure platinumprovides an increased signal representative of glucose concentration ascompared with a signal from a surface region comprising platinizedcarbon.
 9. The transdermal analyte monitoring system of claim 5, whereinthe surface region consisting essentially of pure platinum provides anincreased signal representative of a rate of production of hydrogenperoxide as compared with a signal from a surface region comprisingplatinized carbon.
 10. The transdermal analyte monitoring system ofclaim 1, further comprising an interference filter located between thebiological membrane and the electrode for reducing interference in thetransdermal analyte monitoring system.
 11. The transdermal analytemonitoring system of claim 10, wherein the interference filter comprisesa biocompatible anionic fluoropolymer.
 12. The transdermal analytemonitoring system of claim 1, wherein the biological membrane comprisesskin.
 13. The transdermal analyte monitoring system of claim 1, whereinthe electrode assembly comprises a working electrode, a counterelectrode, and a reference electrode.
 14. A transdermal analytemonitoring system comprising: a medium adapted to interface with abiological membrane and to receive an analyte from the biologicalmembrane; an electrode assembly; and an interference filter locatedbetween the biological membrane and the electrode assembly for reducinginterference from non-target biological moieties in the transdermalanalyte monitoring system.
 15. The transdermal analyte monitoring systemof claim 14, wherein the medium is adapted to react continuously withthe analyte, an electrical signal is detected by the electrode assembly,and the electrical signal correlates to an analyte value.
 16. Thetransdermal analyte monitoring system of claim 14, wherein theinterference filter comprises a biocompatible anionic fluoropolymer. 17.The transdermal analyte monitoring system of claim 14, wherein theinterference filter comprises Nafion.
 18. The transdermal analytemonitoring system of claim 14, wherein the interference filter isselected from the group consisting of:(3-mercaptopropyl)trimethylsilane, cellulose acetate,1,8-diaminonapthaline, phenylenediamine, PTFE, a hydrophobic membrane,Nylon and a hydrophylic membrane.
 19. The transdermal analyte monitoringsystem of claim 14, wherein the interference filter located between theelectrode assembly and the medium.
 20. The transdermal analytemonitoring system of claim 14, where the interference filter is disposedon a surface of an electrode in the electrode assembly.
 21. Thetransdermal analyte monitoring system of claim 14, where theinterference filter is disposed on an outer surface of the electrodeassembly that contacts the biological membrane in operation.
 22. Thetransdermal analyte monitoring system of claim 15, wherein the analytevalue is the flux of the analyte through the biological membrane. 23.The transdermal analyte monitoring system of claim 15, wherein theanalyte value is the concentration of the analyte in a body fluid of asubject.
 24. The transdermal analyte monitoring system of claim 14,wherein the analyte comprises glucose.
 25. The transdermal analytemonitoring system of claim 24, wherein the medium comprises a hydrogeland glucose oxidase.
 26. The transdermal analyte monitoring system ofclaim 14, wherein the interference filter reduces interference effectsfrom unwanted electrochemical oxidation.
 27. The transdermal analytemonitoring system of claim 14, wherein the interference filter reducesan anodic signal produced by electrochemical oxidation of ascorbic acid,uric acide, or acetaminophen.
 28. The transdermal analyte monitoringsystem of claim 14, wherein the interference filter reduces biofouling.29. A method for monitoring an analyte comprising: positioning a mediumwith respect to a biological membrane such that the medium can receivean analyte from the biological membrane, wherein an electrode assemblyis coupled to the medium, the electrode assembly comprises a pluralityof electrodes, and at least one of the electrodes comprises a surfaceregion consisting essentially of pure platinum; continuously reactingthe analyte with the medium; and detecting an electrical signal with theelectrode assembly, wherein the electrical signal correlates to ananalyte value.
 30. The method of claim 29, further comprisingpretreating the biological membrane to increase a permeability of thebiological membrane.
 31. The method of claim 30, wherein the pretreatingstep comprises applying low frequency ultrasound to the biologicalmembrane.
 32. The method of claim 29, wherein the surface regionconsisting essentially of pure platinum provides an increased signalrepresentative of glucose concentration as compared with a signal from asurface region comprising platinized carbon.
 33. The method of claim 29,wherein the surface region consisting essentially of pure platinumprovides an increased signal representative of a rate of production ofhydrogen peroxide as compared with a signal from a surface regioncomprising platinized carbon.
 34. A method for monitoring an analytecomprising: positioning a medium with respect to a biological membranesuch that the medium can receive an analyte from the biologicalmembrane, wherein an electrode assembly is coupled to the medium and aninterference filter is positioned between an electrode in the electrodeassembly and the biological membrane; continuously reacting the analytewith the medium; and detecting an electrical signal with the electrodeassembly, wherein the electrical signal correlates to an analyte value.35. The method of claim 34, further comprising pretreating thebiological membrane to increase a permeability of the biologicalmembrane.
 36. The method of claim 35, wherein the pretreating stepcomprises applying low frequency ultrasound to the biological membrane.37. The method of claim 34, wherein the interference filter comprisesNafion.
 38. The method of claim 34, wherein the interference filter isselected from the group consisting of:(3-mercaptopropyl)trimethylsilane, cellulose acetate,1,8-diaminonapthaline, phenylenediamine, PTFE, a hydrophobic membrane,Nylon and a hydrophylic membrane.
 39. The method of claim 34, whereinthe interference filter reduces interference effects from unwantedelectrochemical oxidation.
 40. The method of claim 34, wherein theinterference filter reduces an anodic signal produced by electrochemicaloxidation of ascorbic acid, uric acide, or acetaminophen.
 41. The methodof claim 34, wherein the interference filter reduces biofouling.